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Optimisation of sample preparation for DNA extraction from formalin fixed paraffin embedded tissues of unresolved sudden unexpected death cases
A retrospective case review revealed an increase in sudden unexpected death (SUD) admittance at Salt River Mortuary (SRM) between 2014 and 2018, and that 40 % of SUD occurred in young individuals between the ages of 1 and 40 years old (SUDY). Despite extensive investigations, the cause of death rema...
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| Format: | Thesis |
| Language: | English |
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Department of Pathology
2021
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| _version_ | 1867613292469420033 |
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| access_status_str | Open Access |
| author | Viljoen, Rabia |
| author2 | Heathfield, Laura |
| author_browse | Heathfield, Laura Viljoen, Rabia |
| author_facet | Heathfield, Laura Viljoen, Rabia |
| author_sort | Viljoen, Rabia |
| collection | Thesis |
| description | A retrospective case review revealed an increase in sudden unexpected death (SUD) admittance at Salt River Mortuary (SRM) between 2014 and 2018, and that 40 % of SUD occurred in young individuals between the ages of 1 and 40 years old (SUDY). Despite extensive investigations, the cause of death remained undetermined in 26 % of SUDY cases. These dormant cases may benefit from retrospective post-mortem molecular autopsies for investigation into genetic causes of death. Often, formalin fixed paraffin embedded tissues (FFPETs) are the only archival sources of DNA available for retrospective analyses. This study aimed to optimise DNA recovery from FFPETs for potential use in molecular autopsies of unresolved SUDY cases. To this end, DNA was extracted from FFPET sections using the QIAamp® DNA FFPE tissue kit; the thickness and number of sections were varied. DNA was assessed using spectrophotometry, real-time PCR and digital capillary electrophoresis. Results showed that finer sectioning (1-µm thick as compared to 3-µm and 5-µm thick), improved DNA concentrations, purities and DNA fragment lengths. Increasing the number of 1-µm thick sections from 30 to 100, significantly improved DNA yield. DNA was not significantly more degraded for FFPETs stored for up to three years, which holds promise in the effectiveness of the technique for aged samples. The DNA extraction method developed in this study yielded a median of 320 ng (287 ng - 698 ng) of DNA with 55 % of DNA fragments being at least 400 bp in size. These results are especially informative for downstream molecular analyses, indicating that genotyping or sequencing assays need to be designed to target amplicons less than 400 bp in size. The degraded nature of the FFPET samples also suggests that massively parallel sequencing might be suited for downstream molecular analysis for determining cause of death in unresolved SUDY cases. |
| format | Thesis |
| id | oai:open.uct.ac.za:11427/33072 |
| institution | University of Cape Town (South Africa) |
| language | eng |
| last_indexed | 2026-06-10T12:33:49.949Z |
| license_str | Not specified — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UCTD — University of Cape Town Open Access Repository |
| publishDate | 2021 |
| publishDateRange | 2021 |
| publishDateSort | 2021 |
| publisher | Department of Pathology |
| publisherStr | Department of Pathology |
| record_format | dspace |
| source_str | UCTD — University of Cape Town Open Access Repository |
| spelling | oai:open.uct.ac.za:11427/33072 Optimisation of sample preparation for DNA extraction from formalin fixed paraffin embedded tissues of unresolved sudden unexpected death cases Viljoen, Rabia Heathfield, Laura Mole, Calvin Biomedical Forensic Science A retrospective case review revealed an increase in sudden unexpected death (SUD) admittance at Salt River Mortuary (SRM) between 2014 and 2018, and that 40 % of SUD occurred in young individuals between the ages of 1 and 40 years old (SUDY). Despite extensive investigations, the cause of death remained undetermined in 26 % of SUDY cases. These dormant cases may benefit from retrospective post-mortem molecular autopsies for investigation into genetic causes of death. Often, formalin fixed paraffin embedded tissues (FFPETs) are the only archival sources of DNA available for retrospective analyses. This study aimed to optimise DNA recovery from FFPETs for potential use in molecular autopsies of unresolved SUDY cases. To this end, DNA was extracted from FFPET sections using the QIAamp® DNA FFPE tissue kit; the thickness and number of sections were varied. DNA was assessed using spectrophotometry, real-time PCR and digital capillary electrophoresis. Results showed that finer sectioning (1-µm thick as compared to 3-µm and 5-µm thick), improved DNA concentrations, purities and DNA fragment lengths. Increasing the number of 1-µm thick sections from 30 to 100, significantly improved DNA yield. DNA was not significantly more degraded for FFPETs stored for up to three years, which holds promise in the effectiveness of the technique for aged samples. The DNA extraction method developed in this study yielded a median of 320 ng (287 ng - 698 ng) of DNA with 55 % of DNA fragments being at least 400 bp in size. These results are especially informative for downstream molecular analyses, indicating that genotyping or sequencing assays need to be designed to target amplicons less than 400 bp in size. The degraded nature of the FFPET samples also suggests that massively parallel sequencing might be suited for downstream molecular analysis for determining cause of death in unresolved SUDY cases. 2021-03-03T01:00:14Z 2021-03-03T01:00:14Z 2020 2021-03-02T18:31:44Z Master Thesis Masters MPhil http://hdl.handle.net/11427/33072 eng application/pdf Department of Pathology Faculty of Health Sciences |
| spellingShingle | Biomedical Forensic Science Viljoen, Rabia Optimisation of sample preparation for DNA extraction from formalin fixed paraffin embedded tissues of unresolved sudden unexpected death cases |
| thesis_degree_str | Master's |
| title | Optimisation of sample preparation for DNA extraction from formalin fixed paraffin embedded tissues of unresolved sudden unexpected death cases |
| title_full | Optimisation of sample preparation for DNA extraction from formalin fixed paraffin embedded tissues of unresolved sudden unexpected death cases |
| title_fullStr | Optimisation of sample preparation for DNA extraction from formalin fixed paraffin embedded tissues of unresolved sudden unexpected death cases |
| title_full_unstemmed | Optimisation of sample preparation for DNA extraction from formalin fixed paraffin embedded tissues of unresolved sudden unexpected death cases |
| title_short | Optimisation of sample preparation for DNA extraction from formalin fixed paraffin embedded tissues of unresolved sudden unexpected death cases |
| title_sort | optimisation of sample preparation for dna extraction from formalin fixed paraffin embedded tissues of unresolved sudden unexpected death cases |
| topic | Biomedical Forensic Science |
| url | http://hdl.handle.net/11427/33072 |
| work_keys_str_mv | AT viljoenrabia optimisationofsamplepreparationfordnaextractionfromformalinfixedparaffinembeddedtissuesofunresolvedsuddenunexpecteddeathcases |